Project Summary How neuronal progenitor cells produce an enormous diversity of neuronal and glial cell types is a fundamental topic that remains largely unresolved. Drosophila has been a pivotal model system to study these complex questions of neurogenesis and research in its nervous system has contributed to several important concepts that apply to mammals. These include temporal and spatial patterning, cell death, neural and/or glial specification and asymmetric cell division. Nevertheless, the full resolution of its neuronal lineages remains elusive. In this proposal we will take advantage of powerful genetic tools derived from synthetic biology to reconstruct the entire neuronal lineage of multiple brain structures at single cell resolution. We will develop transgenic flies in which lineages can be autonomously recorded and analyzed through single cell transcriptomics. We hypothesize that our knowledge on the structure and molecular nature of adult brain development will provide us with a unique advantage to not only reconstruct the entire lineage tree of the Drosophila brain, but also to form new hypotheses on how each of the brain structures form very faithfully and uniformly from precursor cells. Aim 1 Development of a progressive lineage recorder in Drosophila. There are currently no established CRISPR-based lineage methods in the Drosophila nervous system. We will adapt GESTALT in Drosophila to `scar' the DNA during lineage progression and progressively record this lineage through development. We will use genetic tools to gain spatio-temporal control over our lineage measurements and use in silico modeling of the barcode structure to optimize the activity of the system. We will generate flies with enough target sites to capture the entire neuronal diversity generated during neuronal development. We will empirically evaluate different versions of the technology and select the best one for single cell lineage tracing. Aim 2 Defining neuronal birth order and clonal relationships in the adult brain. We will lineage trace through the neuronal development while simultaneous sequencing the transcriptome of single cells. This should allow us to identify the different neural subtypes using our single cell atlas. We will characterize the lineage information per cell and combine this with the published methods capable of reconstructing multi-tree lineages to identify different lineage relationships in our data. We will build on the stereotypical mode of neuronal development to refine this structure and reconstruct the neuronal lineages Aim 3 Experimental validation of the reconstructed neuronal lineages. We will use our prior knowledge of neuronal development in combination with post hoc validation to benchmark our lineage reconstruction. We will first compare our lineage reconstruction of the local motion detectors in Drosophila to their known simple and well-defined lineage. We will use region-specific Gal4 lines to lineage trace different subregions of the neuroepithelium followed by FACS and single cell sequencing to identify the neurons born in these regions. Evaluation of those relationships in our reconstructed tree will provide further validation for our lineage trees.